JPS5934886A - Immobilization of enzyme to surface of porous particle - Google Patents

Abstract

PURPOSE:To prepare immobilized enzyme consisting of an enzyme immobilized firmly to the surface of a porous particle, by coating the surface of a porous particle with a mixture of an aqueous solution of an enzyme and a polymerizable vinyl monomer giving an amorphous polymer, and irradiating the particle with ionizing radiation. CONSTITUTION:A proper amount of a mixture of (A) one or more polymerizable vinyl monomers giving amorphous polymers, such as hydroxyethyl methacrylate, diethylene glycol methacrylate, glycidyl acrylate, etc. and (B) an aqueous solution of an enzyme is applied to the surface of a porous particle having a pore size of preferably 50-5000Angstrom and particle diameter of about 0.5-5mm., e.g. by spraying, immersion, etc. and irradiated with ionizing radiation at 0--100 deg.C at a rate of 10<2>-10<6>rad/hr and total dose of 10<4>-10<6>rad to polymerize the polymerizable vinyl monomer.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for immobilizing enzymes on the surface of porous particles. More specifically, the present invention involves coating the surfaces of porous particles with a mixture consisting of an aqueous enzyme solution and a vitrifiable vinyl polymerizable monomer, and then irradiating the particles with release radiation to fix the enzyme on the particle surface. The present invention relates to a method for immobilizing an enzyme on the surface of porous particles.

The immobilized enzyme produced by the method of the present invention is referred to as "5-porous immobilized enzyme particle".

Pharmaceutical products that use enzymes to carry out useful reactions.

The pharmaceutical and food industry, which produces foodstuffs, has been steadily developing in recent years. However, in the conventional way of using enzymes, the enzyme is dissolved in water and the reaction is carried out in a solution phase, and after the reaction is completed, the enzyme is discharged from the system as waste liquid, so the enzyme reaction process is a batch process. Therefore, the enzyme utilization efficiency was extremely low.

Therefore, in recent years, research has been carried out on so-called enzyme immobilization, in which enzymes are bound or dispersed in polymeric substances, formed into porous gels or powders, and subjected to enzyme reactions as complexes that can be used repeatedly and continuously. has become actively carried out.

Enzyme immobilization has been actively researched since the 1960s and is currently being actively pursued in various countries. An "immobilized enzyme" is an enzyme that is confined within a certain space, performs enzymatic reactions continuously, and can be recovered and reused after the reaction. are classified into comprehensive enzymes and combined enzymes. The former is the comprehensive law (
The latter is produced by the carrier binding method (a method in which enzymes are wrapped and confined in a carrier such as a water-insoluble polymer), and the latter is produced by a carrier binding method (
It is produced by chemically bonding or adsorbing an enzyme to a water-insoluble carrier through covalent or ionic bonds.

The most important issue in immobilization technology is how to maintain the activity of the original enzyme and its sustainability (stability).
The question is whether to fix it so that it is large. Carrier bonding methods, particularly covalent bonding methods, are being enthusiastically attempted because of their long-lasting immobilization effect, but it is said that it is difficult to set the immobilization conditions. On the other hand, the entrapment method physically confines the enzyme, so if the structure of the carrier is devised, it is not difficult to maintain relatively high activity and firmly immobilize the enzyme.

Another attractive feature of the comprehensive method is that it is possible to enhance the activity by including effective auxiliary components other than enzymes together with the enzyme, and to immobilize relatively complex enzyme reaction systems consisting of multiple components as they are. As entrapment methods, the lattice type and the microcapsule type are known, and these methods confine the enzyme in a network-structured polymer gel and polymer microcapsule, respectively. However, the conventional entrapment method has the drawback that the enzyme is largely detached from the carrier, and it has also been considered impossible to immobilize the enzyme on the particle surface of the carrier. Immobilizing an enzyme on the surface of a carrier is a necessary condition for carrying out enzymatic reactions using immobilized enzymes with large molecular weight substrates, and even in the case of enzymatic reactions with low molecular weight substrates, the diffusion law is maintained. Since the problem of regulation can be ignored, the reaction can proceed efficiently.

In order to prevent the enzyme from detaching from the carrier, the present inventors
proposed a method for producing a composition by uniformly mixing a porous adsorbent, a polymerizable monomer, and an aqueous enzyme or bacterial cell solution, and irradiating the mixture with radiation (Japanese Patent Publication No. 56-5398
8). In the case of this conventional method, the porous adsorbent is powdered and mixed with a monomer or enzyme aqueous solution, the entire system is solidified by polymerization, and after polymerization, the immobilized material is crushed to obtain an immobilized material of a certain size. It's a method. This conventional method has the disadvantage that it is impossible to immobilize the enzyme on the particle surface from the beginning, resulting in a diffusion-controlled immobilization product, which cannot be used for enzymatic reactions with high-molecular-weight substrates. Therefore, the present inventors have conducted repeated research on a method for easily obtaining particulate immobilized substances with a large surface area without pulverization.
The present invention was completed by discovering a method for firmly immobilizing enzymes on the surface of porous particles.

Therefore, the main object of the present invention is to provide an immobilized enzyme in which the enzyme is firmly immobilized on the surface of porous particles, and a method for producing the same.

A more specific object of the present invention is to coat the surfaces of porous particles with a mixture consisting of an aqueous enzyme solution and a vitrifiable vinyl polymerizable monomer, and then irradiate the porous particles with ionizing radiation to remove the polymerizable monomer. An object of the present invention is to provide a method for immobilizing an enzyme on the surface of a porous particle, which comprises immobilizing the enzyme on the particle surface by polymerizing on the particle surface.

Other objects and advantages of the invention will be successively identified below.

Clarifying the structure of the present invention: According to the present invention, an aqueous enzyme solution and a vitrifiable vinyl polymerizable monomer are mixed, and after coating the surfaces of selected porous particles, the mixture is heated to 0°C. The enzyme immobilized on the surface of the porous particles is maintained at a low temperature of -100°C and irradiated with ionizing radiation to polymerize the polymerizable monomer and immobilize the enzyme on the surface of the porous particles. Manufactured.

One of the features of the present invention is that a vitrifiable vinyl polymerizable monomer is used as the polymerizable monomer. The vitrifying polymerizable monomer is a polymerizable monomer that does not crystallize at low temperatures but becomes supercooled and does not lose its polymerizability. Polymerization of vitrifying polymerizable monomers in a supercooled state can be called solid-phase polymerization in an amorphous state, and since the polymerizability is high in a low-temperature region, it can be used for low-temperature polymerization such as immobilization of living organisms. This is an extremely useful tool for applications. Since enzymes are usually thermally unstable, it is ideal to immobilize at a lower temperature in order to prevent deactivation of enzyme activity.

The radiation polymerization method is a method that can effectively carry out polymerization at low temperatures, and considering that vitrifiable vinyl polymerizable monomers have high polymerizability at low temperatures by radiation, the present invention It can be said that this was achieved by using a vitrifying polymerizable monomer. By the way, vitrifying polymerizable monomers contain hydrogen-bonding functional groups with moderate strength, bulky or significantly asymmetric substituents,
It contains a bond with low rotational free energy such as an ether bond, and one or more of those represented by the following general formula are used; a, CH2=CX-C=O (CH2)nOR+ where X is H or a methyl group, R1 is H or CH2=CX-C- where X is H or a methyl group, and n is an integer from 4 to 1o; b, CH2=cx c-o (R2)m Cc
x=cn20 0 where X is H or a methyl group, and horse is -CH2CH20-.

-CH-CH20- or -CH2-CHO-.

1 H3cH3 and m is an integer from 1 to 3; where X is H or a methyl group; d, CH2=CX COR3() R4 where X is H or a methyl group, R3 is a straight chain having 1 to 10 carbon atoms; a chain or branched alkylene group, and R4 is a vinyl or alkyl group having 1 to 10 carbon atoms; 1\OR7 where R3 is an alkane(01-C5)-yl-isido.

alkylene (C,=C5) amino group % R6 and H are each H, an alkyl group having 1 to 5 carbon atoms, 1 to
Alkylamino groups with 5 carbon atoms, hydroxylalkyl groups with 1 to 5 carbon atoms.

Allyl or vinyl group; f=CH2=CX-C-R30R80-R4 where X
, R3 and R4 are as defined above, R8 is the same as R3, R3 and R8 are the same and each different; where X
, R3 and R4 are as defined above; where X, R3
and mountains as defined above; where X is as defined above and R1□ is benzyl, tolyl, xylyl,
Phenyl, furfuryl.

Naphthyl, phthalyl, cyclohexyl, cyclophenyl, cycloheptyl, cyclobutyl, pyridyl or ro-oxopyrrolidinyl group; 8R9C(CH20CCX=CH2)! "where X is as defined above and R-ethyl or propyl group; or where Branched alkylene group, 10HRu where R11 is as defined above.

Specific examples of the vitrifiable vinyl polymerizable monomers used in the present invention include: hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxypropyl acrylate, hydroxypropyl methacrylate, diethylene glycol methacrylate, diethylene glycol acrylate, triethylene glycol methacrylate. , triethylene glycol acrylate, tetraethylene glycol acrylate, tetraethylene glycol methacrylate, polyethylene glycol methacrylate, polyethylene glycol acrylate,
Diethylene glycol dimethacrylate, diethylene glycol diacrylate, methoxydiethylene glycol dimethacrylate, methoxydiethylene glycol diacrylate, methoxytetraethylene glycol dimethacrylate, neopentyl glycol dimethacrylate, hexanediol monomethacrylate.

glycidyl methacrylate, glycidyl acrylate,
Examples include heptanediol monomethacrylate, butanediol monomethacrylate, ethylene glycol diacrylate, and propylene glycol dimethacrylate.

The porous particles used in the present invention include alumina particles, activated carbon particles, molecular annealing particles, etc., but the pore diameter of these porous particles is preferably 50 to 5,000. mm z is preferred. There are various materials that can be used as porous materials, but the porous particles used in the present invention do not act as mere adsorbents, but act as porous surface substrates that allow enzyme molecules to adhere to the surface, so the larger the pore size, the better. In addition, unless the porous particles can withstand a certain degree of mechanical strength, they cannot be used for continuous reactions by filling a reaction vessel with immobilized enzyme particles and stirring them.

Therefore, alumina particles and the like are suitable in these respects.

Examples of enzymes that have particularly remarkable effects of the present invention and are suitable for applying the method of the present invention include glucoamylase, amylase, cellulase, hemicellulase, β-glucosidase, glucose imerase, trypsin, chymotrypsin, and dino2- enzyme, uricase, esterase, glucose oxidase, etc.

In the present invention, the surface of the porous particles may be coated with the aqueous solution of the enzyme and the mixture of the polymerizable monomer by any method such as spraying or immersing the porous particles in the mixture. It is important to coat the surface of the particles thinly. The simplest coating method is to place the porous particles in a container and gradually add a mixture of the enzyme aqueous solution and the monomer while stirring. The important point in this coating operation is not to add an excessive amount of the mixture of enzyme and monomer aqueous solution.

In the present invention, porous particles are uniformly coated with an appropriate amount of a mixture of an enzyme and a polymerizable monomer, and then rapidly cooled to a low temperature of 0°C to -100°C to solidify the coating.

By irradiating the porous particles with ionizing radiation in this state, it is possible to produce particles in which enzymes are immobilized on the surface of the porous particles. In order to expose the enzyme as much as possible on the surface of the porous particles, the enzyme or its aqueous solution may be coated on the surface of the porous particles, which have been previously coated with a polymerizable monomer at a certain concentration. In this way, the method of coating the surface of porous particles with a thin aqueous solution of an enzyme and a polymerizable monomer and rapidly polymerizing the enzyme to immobilize the enzyme on the surface of the porous particles is only possible with the low-temperature radiation polymerization method; is an important feature of the invention.

The ionizing radiation used in the present invention is alpha rays and beta rays.

γ-rays, X-rays, and electron beams, and the usable dose is 104~
It is in the range of 10' rad. Incidentally, the effect of radiation irradiation on enzyme activity is significantly dependent on the irradiation temperature and dose, and deactivation due to irradiation increases rapidly under irradiation conditions of 0° C. or higher and 106 rad or higher. On the contrary, as long as the reaction is carried out quickly under irradiation conditions of 10' to 10' rad in the temperature range of D°C to -100°C, almost no effect of irradiation on enzyme activity is observed.

The porous immobilized enzyme particles of the present invention have the following advantages and characteristics.

(a) The enzyme can be used repeatedly and continuously for a long period of time, and the large surface area makes it easy to handle at 0°C. (b) The enzyme is immobilized on the particle surface, so it can also be used for enzyme reactions with water-insoluble substrates. I can do it.

Example 1 5 parts of 0.5 glucoamylase aqueous solution and 5 parts of hydroxymethyl methacrylate were mixed, and this was mixed into a diameter of 2 to 6 mm.
Add it little by little while stirring to a container containing 600 parts of alumina. After addition, mix the inside of the container well to uniformly coat the surface of the particles with the enzyme monomer aqueous solution.
Cooled to ℃. In this state, the γ-ray is I x 10' r/
Irradiation was performed for 1 hour at a dose rate of hr. After irradiation, the container was brought to room temperature and the contents inside the container were taken out to obtain immobilized enzyme particles immobilized on the particle surface. The particles were air-dried at room temperature and then used in an enzyme reaction. A column was filled with 100 g of the obtained particles, and a continuous enzymatic reaction was performed using maltose as a substrate. When the enzymatic reaction was continued at 60° C. for one month at a flow rate of 2 cc/hr, the yield of glucose production remained constant at 95 cm.

Example 2 6 parts of 5-cellulase aqueous solution and 4 parts of hydroxypropyl acrylate were mixed, and this was mixed with alumina 4 with a diameter of 2 to 6 mm.
Add it little by little while stirring to the container containing 00 parts.
After coating in the same way as in Example 1, γ at a temperature of -78 °C
The radiation was applied for 1 hour at a dose rate of I x 10' rad/hr. A batch enzymatic reaction was carried out with the thus obtained immobilized cellulase particles using an aqueous solution of cellulose powder 5T of Roro 00 mesh, which is a water-insoluble substrate, as a substrate. That is, particles 20.9 and 5 polycellulose aqueous solution funnel
1 was placed in a 100 ml Erlenmeyer flask and subjected to batch enzymatic reaction for 48 hours with shaking at 40°C. As a result, the glucose production rate remained constant at 60% during the batch reaction for one month.

Example 6 parts of 0.5% β-glucosidase aqueous solution and 7 parts of hydroxyethyl acrylate were mixed, and this was mixed into a 1-4 mm diameter
The activated carbon particles were added little by little with stirring to a container containing activated carbon particles, and after the particles were well coated, the mixture was cooled to -24°C and β rays were absorbed at a dose rate of 5 x IQ” rad/hr.
Irradiated for hours. After polymerization, the particles were air-dried and then subjected to continuous enzymatic reactions using cellbiose as a substrate. A column was packed with 50 g of immobilized β-glucosidase particles, and a 5-cell cellbiose aqueous solution was passed through the column at a flow rate of 3 cc/hr.
Continuous enzymatic reaction was carried out for several months, but the glucose production rate was 8.
It remained constant at 0% and did not change.

Patent applicant: Japan Atomic Energy Research Institute (2 others)

Claims (1)

[Claims] 1. A mixture of an aqueous enzyme solution and one or more vitrifying vinyl polymerizable monomers is coated on the surface of porous particles, and then kept at a low temperature of 0°C to -100°C. ionizing radiation at a dose rate of 102 to 10' rad/hr.
A method for firmly immobilizing enzymes on the surface of porous particles, which consists of irradiating at 4 to 10 degrees. 2. Porous particles have a pore size of 5U, 5,0OOA, and a diameter of 0.5
Claim 1, characterized in that the diameter is 5 mm.
The method described in section. 6. The method according to claim 81, wherein the vitrifiable vinyl polymerizable monomer is one or more selected from the following general formula. a, CH2=CX C0(CH2) no & where X is H
or methyl group, R1 is H or CH2=CX-C- 1 0 (1) where X is ■ (or methyl group, and n is an integer from 4 to 10; CL CI (20
−. m is an integer from 1 to O; where X is H or a methyl group; d, CH2=CX-C-0-R3-0-R. 1 where X is H or a methyl group I R3 is a straight or branched alkylene group having 1 to 10 carbon atoms. and R4 is a vinyl or alkyl group having 1 to 10 carbon atoms; where the island is an alkane (C□~C,)-IruiV(2) de, an alkane (C1~C3) amine group, &O and R7
are each H, an alkyl group having 1 to 5 carbon atoms, 1
alkylamino group with ~5 carbon atoms, hydroxylalkyl group with 1 to 5 carbon atoms, allyl or vinyl group; f, CH2=CX CR30R80R4
where X, R3 and R4 are as defined above, R8 is the same as horse, R3 and R8 are the same and each different; where X, R3 and R4 are as defined above; where X, R3 and R4 as defined above; i, CH
2=CX-C-0-R. 1 where X is as defined above and 1 (11 is a benzyl, tolyl, xylyl, phenyl, furfuryl, naphthyl, phthalyl, cyclohexyl; cyclophenyl, cycloheptyl, cyclobutyl, pyridyl or 6-oxopyrrolidinyl group; 8R, -C-(C
H2-0-C-CX=CH2)31 where X is as defined above and is an ethyl or propyl group; or where X is as defined above and Rlo is isopropylene, isobutylene, 1- a branched alkylene group having 5 carbon atoms, where R11 is as defined above.